Bacteria communicate with each other to coordinate expression of specific genes in a cell density dependent fashion. This “bacterial signaling” is a phenomenon called quorum sensing and response. Quorum sensing enables a bacterial species to sense its own number and regulate gene expression according to population density. In other words, quorum sensing is cell density-dependent regulation of genes that involves a freely diffusible molecule synthesized by the cell called an autoinducer (Fuqua, W. C. et al. (1996) Annu. Rev. Microbiol. 50:727-751; Salmond, G. P. C. et al. (1995) Mol. Microbiol. 16:615-624; Sitnikov, D. M. et al. (1995) Mol. Microbiol. 17:801-812). Autoinducers are described, e.g., in U.S. Pat. Nos. 5,591,872 and 5,593,827.
Autoinducer molecules and methods for the use of autoinducer molecules are described, for example, in U.S. Pat. Nos. 5,591,872 and 6,057,288, and in published PCT international patent application Nos. WO 98/57618, WO 98/58075, WO 99/65889, and WO 00/06177. Bacteria at a low cell density produce a basal level of autoinducer, and, as a population grows, autoinducer concentration increases concomitantly with cell density. On reaching a threshold concentration, autoinducer binds to and thereby activates an R protein, which then induces or ceases to repress specific target genes. In this manner, intercellular signals enable a bacterial population to control the expression of specific genes in response to cell density.
The paradigm system for quorum sensing is the lux system of the luminescent marine bacterium, Vibrio fischeri. V. fischeri exists at low cell densities in sea water and also at very high cell densities within the light organs of various marine organisms, such as the squid Euprymna scolopes (Pesci, E. C. et al. (1997) Trends in Microbiol. 5(4):132-135; Pesci, E. C. et al. (1997) J. Bacteriol. 179:3127-3132; Ruby, E. G. (1996) Ann. Rev. Microbiol. 50:591-624). At high cell densities, the V. fischeri genes encoding the enzymes required for light production are expressed. These genes are part of the lux ICDABEG operon and are regulated by the gene products of luxI and luxR (Baldwin, T. O. et al. (1989) J. of Biolum. and Chemilum. 4:326-341; Eberhard, A., et al. (1991) Arch. of Microbiol. 155:294-297; Gray, K. M. et al. (1992) J. Bacteriol. 174:4384-4390).
LuxI is an autoinducer synthase that catalyzes the formation of the V. fischeri autoinducer (VAI), N-(3-oxohexanoyl) homoserine lactone (Eberhard, A., et al. (1991) Arch. of Microbiol. 155:294-297; Seed, P. C. et al. (1995) J. Bacteriol. 177:654-659). The autoinducer freely diffuses across the cell membrane and at high cell densities, reaches a critical concentration (Kaplan, H. B. et al. (1985) J. Bacteriol. 163:1210-1214). At this critical concentration, VAI interacts with LuxR, a DNA-binding transcriptional regulator. The LuxR-VAI complex then binds to an upstream sequence of the lux operon called the “lux box”, and activates transcription (Devine, J. H. et al. (1989) PNAS 86: 5688-5692; Hanzelka, B. A. et al. (1995) J Bacteriol. 177:815-817; Stevens, A. M. et al. (1994) PNAS 91:12619-12623). Since one of the genes of the operon is luxI, an autoregulatory loop is formed.
Many gram-negative bacteria have been shown to possess one or more quorum sensing systems (Fuqua, W. C. et al. (1996). Annu. Rev. Microbiol. 50:727-751; Salmond, G. P. C. et al. (1995) Mol. Microbiol. 16:615-624). These systems regulate a variety of physiological processes, including the activation of virulence genes. In addition, it has been recently demonstrated that quorum sensing is involved in biofilm formation (Davies, D. G. et al. (1998) Science. 280(5361):295-8).
The systems typically have acylated homoserine lactone (“HSL”) ring autoinducers, in which the homoserine lactone ring is conserved. The acyl side chain, however, can vary in length and degree of substitution. Pseudomonas aeruginosa has two quorum sensing systems, las and rhl (Brint et al. 1995, Hanzelka et al. 1996, Baldwin et al., 1989, Passador et al. 1993, Pearson et al. 1997, Pesci et al. 1997). The two systems have distinct autoinducer synthases (lasI and rhlI), transcriptional regulators (lasR and rhlR), and autoinducers (N-(3-oxododecanoyl) homoserine lactone (HSL) and N-butyryl HSL) (Sitnikov et al., 1995, Stevens et al. 1994). N-(3-oxododecanoyl) homoserine lactone is synthesized by LasI along with a small amount of N-(3-oxooctanoyl) HSL and N-(3-oxohexanoyl) HSL, while RhlI makes primarily N-butyryl HSL and a small amount of N-hexanoyl (Pearson et al. 1994, Winson et al. 1995). The rhl and las systems are involved in regulating the expression of a number of secreted virulence factors, biofilm development, and the stationary phase sigma factor (RpoS) (Brint et al. 1995, Davies et al. 1998, Latifi et al. 1996, Ochsner et al. 1995, Pesci et al. 1997). Expression of the rhl system requires a functional las system. Therefore the two systems in combination with RpoS constitute a regulatory cascade (Pesci et al. 1997, Seed et al. 1995).
Biofilms are defined as an association of microorganisms, single or multiple species, that grow attached to a surface and produce a slime layer that provides a protective environment (Costerton, J. W. (1995) J Ind Microbiol. 15(3): 137-40, Costerton, J. W. et al. (1995) Annu Rev Microbiol. 49:711-45). Typically, biofilms produce large amounts of extracellular polysaccharides, responsible for the slimy appearance, and are characterized by an increased resistance to antibiotics (1000- to 1500-fold less susceptible). Several mechanisms are proposed to explain this biofilm resistance to antimicrobial agents (Costerton, J. W. et al. (1999) Science. 284(5418):1318-22). One idea is that the extracellular matrix in which the bacterial cells are embedded provides a barrier toward penetration by the biocides. A further possibility is that a majority of the cells in a biofilm are in a slow-growing, nutrient-starved state, and therefore not as susceptible to the effects of antimicrobial agents. A third mechanism of resistance could be that the cells in a biofilm adopt a distinct and protected biofilm phenotype, e.g., by elevated expression of drug-efflux pumps.
In most natural settings, bacteria grow predominantly in biofilms. Biofilms of P. aeruginosa have been isolated from medical implants, such as indwelling urethral, venous or peritoneal catheters (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90). Chronic P. aeruginosa infections in cystic fibrosis lungs are considered to be biofilms (Costerton, J. W. et al. (1999) Science. 284(5418): 1318-22).
In industrial settings, the formation of biofilms is often referred to as ‘biofouling’, or biological fouling. Biological fouling of surfaces is common and leads to material degradation, product contamination, mechanical blockage, and impedance of heat transfer in water-processing systems. Biofilms are also the primary cause of biological contamination of drinking water distribution systems, due to growth on filtration devices.
As noted earlier, many gram-negative bacteria have been shown to possess one or more quorum sensing systems that regulate a variety of physiological processes, including the activation of virulence genes and biofilm formation. One such gram negative bacterium is Pseudomonas aeruginosa. 
P. aeruginosa is a soil and water bacterium that can infect animal hosts. Normally, the host defense system is adequate to prevent infection. However, in immunocompromised individuals (such as burn patients, patients with cystic fibrosis, or patients undergoing immunosuppressive therapy), P. aeruginosa is an opportunistic pathogen, and infection with P. aeruginosa can be fatal (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74; Van Delden, C. et al. (1998) Emerg Infect Dis. 4(4):551-60).
For example, Cystic fibrosis (CF), the most common inherited lethal disorder in Caucasian populations (˜1 out of 2,500 life births), is characterized by bacterial colonization and chronic infections of the lungs. The most prominent bacterium in these infections is P. aeruginosa—by their mid-twenties, over 80% of people with CF have P. aeruginosa in their lungs (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74). Although these infections can be controlled for many years by antibiotics, ultimately they “progress to mucoidy,” meaning that the P. aeruginosa forms a biofilm that is resistant to antibiotic treatment. At this point the prognosis is poor. The median survival age for people with CF is the late 20s, with P. aeruginosa being the leading cause of death (Govan, J. R. et al. (1996) Microbiol Rev. 60(3):539-74). According to the Cystic Fibrosis Foundation, treatment of CF cost more than $900 million in 1995 (Foundation, CF http://www.cff.org/homeline199701.htm).
P. aeruginosa is also one of several opportunistic pathogens that infect people with AIDS, and is the main cause of bacteremia (bacterial infection of the blood) and pneumonitis in these patients (Rolston, K. V. et al. (1990) Cancer Detect Prev. 14(3):377-81; Witt, D. J. et al. (1987) Am J. Med. 82(5):900-6). A recent study of 1635 AIDS patients admitted to a French hospital between 1991-1995 documented 41 cases of severe P. aeruginosa infection (Meynard, J. L. et al. (1999) J Infect. 38(3): 176-81). Seventeen of these had bacteremia, which was lethal in 8 cases. Similar numbers were obtained in a smaller study in a New York hospital, where the mortality rate for AIDS patients admitted with P. aeruginosa bacteremia was about 50% (Mendelson, M. H. et al. 1994. Clin Infect Dis. 18(6):886-95).
In addition, about two million Americans suffer serious burns each year, and 10,000-12,000 die from their injuries. The leading cause of death is infection (Lee, J. J. et al. (1990) J Burn Care Rehabil. 11(6):575-80). P. aeruginosa bacteremia occurs in 10% of seriously burned patients, with a mortality rate of 80% (Mayhall, C. G. (1993) p. 614-664, Prevention and control of nosocomial infections. Williams & Wilkins, Baltimore; McManus, A. T et al. (1985) Eur J Clin Microbiol. 4(2):219-23).
Such infections are often acquired in hospitals (“nosocomial infections”) when susceptible patients come into contact with other patients, hospital staff, or equipment. In 1995 there were approximately 2 million incidents of nosocomial infections in the U.S., resulting in 88,000 deaths and an estimated cost of $ 4.5 billion (Weinstein, R. A. (1998) Emerg Infect Dis. 4(3):416-20). Of the AIDS patients mentioned above who died of P. aeruginosa bacteremia, more than half acquired these infections in hospitals (Meynard, J. L. et al. (1999) J Infect. 38(3):176-81).
Nosocomial infections are especially common in patients of intensive care units as these people often have weakened immune systems and are frequently on ventilators and/or catheters. Catheter-associated urinary tract infections are the most common nosocomial infection (Richards, M. J. et al. (1999) Crit Care Med. 27(5):887-92) (31% of the total), and P. aeruginosa is highly associated with biofilm growth and catheter obstruction. While the catheter is in place, these infections are difficult to eliminate (Stickler, D. J. et al. (1998) Appl Environ Microbiol. 64(9):3486-90). The second most frequent nosocomial infection is pneumonia, with P. aeruginosa the cause of infection in 21% of the reported cases (Richards, M. J. et al. (1999) Crit Care Med. 27(5):887-92). The annual costs for diagnosing and treating nosocomial pneumonia has been estimated at greater than $2 billion (Craven, D. E. et al. (1991) Am J. Med. 91(3B):44S-53S).
Treatment of these so-called nosocomial infections is complicated by the fact that bacteria encountered in hospital settings are often resistant to many antibiotics. In June 1998, the National Nosocomial Infections Surveillance (NNIS) System reported increases in resistance of P. aeruginosa isolates from intensive care units of 89% for quinolone resistance and 32% for imipenem resistance compared to the years 1993-1997 (NNIS. http://www.cdc.gov/ncidod/hip/NNIS/AR_Surv1198.htm). In fact, some strains of P. aeruginosa are resistant to over 100 antibiotics (Levy, S. (1998) Scientific American. March). There is a critical need to overcome the emergence of bacterial strains that are resistant to conventional antibiotics (Travis, J. (1994) Science. 264:360-362).
P. aeruginosa is also of great industrial concern (Bitton, G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y.; Steelhammer, J. C. et al. (1995) Indust. Water Treatm.:49-55). The organism grows in an aggregated state, ie., the biofilm, which causes problems in many water processing plants. Of particular public health concern are food processing and water purification plants. Problems include corroded pipes, loss of efficiency in heat exchangers and cooling towers, plugged water injection jets leading to increased hydraulic pressure, and biological contamination of drinking water distribution systems (Bitton, G. (1994) Wastewater Microbiology. Wiley-Liss, New York, N.Y., 9). The elimination of biofilms in industrial equipment has so far been the province of biocides. Biocides, in contrast to antibiotics, are antimicrobials that do not possess high specificity for bacteria, so they are often toxic to humans as well. Biocide sales in the US run at about $ 1 billion per year (Peaff, G. (1994) Chem. Eng. News: 15-23).
A particularly ironic connection between industrial water contamination and public health issues is an outbreak of P. aeruginosa peritonitis that was traced back to contaminated poloxamer-iodine solution, a disinfectant used to treat the peritoneal catheters. P. aeruginosa is commonly found to contaminate distribution pipes and water filters used in plants that manufacture iodine solutions. Once the organism has matured into a biofilm, it becomes protected against the biocidal activity of the iodophor solution. Hence, a common soil organism that is harmless to the healthy population, but causes mechanical problems in industrial settings, ultimately contaminated antibacterial solutions that were used to treat the very people most susceptible to infection.
Regulation of virulence genes by quorum sensing is well documented in P. aeruginosa. Recently, genes not directly involved in virulence including the stationary phase sigma factor rpoS and genes coding for components of the general secretory pathway (xcp) (Jamin, M. et al. (1991) Biochem J. 280(Pt 2):499-506) have been reported to be positively regulated by quorum sensing. Furthermore, the las quorum sensing system is required for maturation of P. aeruginosa biofilms (Chapon-Herve, V. et al. (1997) Mol. Microbiol. 24, 1169-1170; Davies, D. G., et al. (1998) Science 280, 295-298). Thus it seems clear that quorum sensing represents a global gene regulation system in P. aeruginosa. However, the number and types of genes controlled by quorum sensing have not been identified or studied extensively.